WO2020185536A1 - A novel vector for gene transfer and gene copy proliferation - Google Patents

Abstract

The invention relates to a circular vector for plant transformation comprising: a first tethering nucleic acid and a second tethering nucleic acid; a nucleic acid encoding a polynucleotide of interest (POI); a nucleic acid comprising an origin of replication; and two or more nucleic acids encoding replicon proteins and methods for using the same. Also provided are plants produced by the methods of the invention and products produced from the plant.

Description

A NOVEL VECTOR FOR GENE TRANSFER AND GENE COPY PROLIFERATION

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. § 1.821, entitled 9662-71WO_ST25.txt, 577,450 bytes in size, generated on March 5, 2020 and filed via EFS- Web, is provided in lieu of a paper copy. This Sequence Listing is hereby incorporated herein by reference into the specification for its disclosures.

STATEMENT OF PRIORITY

This application claims the benefit, under 35 U.S.C. § 119 (e), of U.S. Provisional Application No. 62/815,628 filed on March 8, 2019, the entire contents of which is incorporated by reference herein.

FIELD

The invention relates to a vector for plant transformation and methods for using the same.

BACKGROUND

Genomic plasticity and adaptation is common to all life forms. A detailed understanding of the complex link between the genome and the phenome that governs adapted traits is one of the grand challenges of biology, from weed science to cancer evolution. Knowledge of the biological mechanisms and regulatory factors of adaptation will be critical in the 21^st century in delivering stable and secure food, fuel, fiber, and health innovations to the growing population.

Gene duplications are perhaps among the oldest and most frequent sources of genetic diversity in all organismal species. Gene duplications are a signature of genomic adaptation and evolution, and can be triggered by selective pressures to endow or enhance phenotypes for organisms to survive and adapt to environmental perturbations (both subtle and extreme). Little is known about the molecular mechanisms by which certain genes amplify and proliferate in the genome.

Extrachromosomal DNAs (eccDNAs) are one form by which genes can become amplified. EccDNAs are an understudied fraction of the genome that are present across

Kingdoms, and in limited reports, have been shown associate with biological functions in the cell, such as, maintenance of genome stability and cell aging, and proliferation of oncogenes that contribute to tumor evolution and genetic heterogeneity in cancers. The presence and conservation of eccDNAs across Kingdoms and aggressive disease states suggests a selective advantage and a fundamental biological role in the cell.

Previous reports of eccDNAs show they can range in size from a few kilobases in plants to nearly 40kb in yeast, with only a small body of insight into how these elements form and exist in the cell. There is little to no evidence that describes how eccDNAs function, persist in the genome, and contribute to genome dynamics and the enhancement of existing traits or presentation of new traits.

Genome engineering through technologies such as Crispr-CAS9 hold the promise to deliver quantum leaps in genetically tailoring plants to ensure food, fuel, and fiber security. However, the current approaches are hindered by both off-target and genome integration difficulties. The present invention overcomes these shortcomings in the art by providing a novel plant based vector for plant genetic engineering.

SUMMARY

One aspect of the invention is a circular plant vector comprising: a first tethering nucleic acid and a second tethering nucleic acid; a nucleic acid encoding a polynucleotide of interest (POI); a nucleic acid comprising an origin of replication; and two or more nucleic acids encoding replicon proteins.

A second aspect of the invention is a method of expressing a polynucleotide of interest in a plant or part thereof, the method comprising introducing into the plant or part thereof the circular plant vector of the invention, and selecting a plant or part thereof expressing the polynucleotide of interest.

An third aspect is a method of modulating the expression a polynucleotide of interest in a plant cell, the method comprising introducing into a plant cell the circular plant vector of the invention to produce a transformed plant cell expressing the polynucleotide of interest.

A further aspect is a method of producing a plant cell expressing a polynucleotide of interest, the method comprising introducing into a plant cell the circular plant vector of the invention, thereby producing a plant cell comprising the polynucleotide of interest.

Also provided are plants, plant parts thereof including cells that comprise the vector of the invention as well as crops comprising the plants of the invention and products produced from the plants of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 provides a cartoon representation of unique tethering mechanism by which the eccDNA can tether to nuclear chromatin through a unique tethering mechanism (panel A) or tethering and nuclear integration into open chromatin (panel B) of a construct of the invention comprising eccDNA.

Fig 2. The eccDNA Replicon. Panel A. The structure of the eccDNA replicon. The blue circular ideogram represents the replicon sequence (SEQ ID NO:20). The multicolored histograms represent the overlapping BAC tile path, the third inner track depicts the 110 putative protein coding gene sequences, those transcribed in the clockwise direction are depicted in green and counterclockwise in yellow. The internal links connect repetitive sequences to their respective internal matches; red (direct) and blue (inverted). Panel B. The eccDNA replicon with the predicted tethering region highlighted from ~95kb to 175kb. The red links are palindromic sequences (elevated A+T content) that may function in attachment to nuclear chromatin. Panel C. Fiber-FISH images of eccDNAs in GR A. palmeri with 80 EPSPS copies. (A) Circular form of eccDNA. (B) Linear form of eccDNA. (C) Dimerized circular form of eccDNA with head-to-tail tandem orientation. (D) Linear form of eccDNA with head-to-tail dimer. (E) Atypical fiber representing structural changes. Note: In the relatively long DNA fibers (D, E), two images were captured sequentially with an overlapping region and then they were combined into a single image using Adobe Photoshop. 1, BAC 01G15; 2, BAC 13C09; 3, BAC 22F22; 4, BAC 23A10; 5, BAC 03A06; 6, BAC 08H14. (Scale bars, 10 pm.). Panel D. Distribution of eccDNAs (red signals) on meiotic chromosomes in microsporocytes of GR A. palmeri during progression from the leptotene stage of meiosis I through anaphase of meiosis II (A-I) and pollen (J) detected by FISH (arrowheads point to the eccDNAs that are not associated with chromosomes). Brackets in G and I represent the lagging eccDNAs associated with chromatin bridges at anaphase to telophase stages.

Fig. 3. GC/AT map of the eccDNA replicon, and origin of replication. Panel A.

G+C/A+T map of the plant eccDNA replicon. The extended autonomous consensus sequence (EACS) region is highlighted in transparent yellow and serves as the origin of replication of the eccDNA replicon. Panel B. Top and bottom: A zoomed region of the DNA unwinding element and the extended autonomous consensus sequence regions that function as the origin of replication. Fig. 4. Repeat structure of the eccDNA replicon. Panel A. circos plot of the Amaranthus palmeri eccDNA replicon and key repetitive structure. The outer colored histograms with labels are predicted repetitive elements with interesting arrangements. The highlighted repeat arrays are the clustered long and short interspersed palindromic repeat (CLiSPr) arrays that flank the EPSPS synthase gene. These repeat blocks are larger, asymmetric direct repeats. The inner tracks depict predicted MITE and helitron repetitive elements. The internal red links are direct repeats and their relationships within the replicon. Panel B.

Everything in Panel A, except the direct repeats are hidden and inverted repeats are illustrated in blue.

DETAILED DESCRIPTION

The present invention now will be described more fully hereinafter with reference to the accompanying drawings and examples, in which embodiments of the invention are shown. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention.

For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. Thus, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention. Hence, the following descriptions are intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and/or paragraph in which the reference is presented. In case of a conflict in terminology, the present specification is controlling.

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

It will be understood that, although the terms "first," "second," etc. may be used herein to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element from another. Thus, a "first" element discussed below could also be termed a "second" element without departing from the teachings of the present invention. The sequence of operations (or steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.

As used in the description of the invention and the appended claims, the singular forms “a,”“an” and“the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Also as used herein,“and/or” refers to and encompasses any and all possible

combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

The term“about,” as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example, “about X” where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measureable value may include any other range and/or individual value therein.

As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y" and phrases such as "from about X to Y" mean "from about X to about Y."

The term“comprise,”“comprises” and“comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase“consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term“consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to“comprising.”

It will also be understood that, as used herein, the terms "example," "exemplary," and grammatical variations thereof are intended to refer to non-limiting examples and/or variant embodiments discussed herein, and are not intended to indicate preference for one or more embodiments discussed herein compared to one or more other embodiments.

As used herein, the terms“increase,”“increasing,”“increased,”“enhance,”

“enhanced,”“enhancing,” and“enhancement” (and grammatical variations thereof) describe an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control.

As used herein, the terms“reduce,”“reduced,”“reducing,”“reduction,”“diminish,” and“decrease” (and grammatical variations thereof), describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control. In particular embodiments, the reduction can result in no or essentially no (i. e. , an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.

A“heterologous” or a“recombinant” nucleic acid is a polynucleotide sequence not naturally associated with a host cell into which it is introduced, including non- naturally occurring multiple copies of a naturally occurring nucleotide sequence. As another example, the circular plant vector of the invention is a recombinant vector because it is non-naturally occurring and is therefore heterologous to any plant host into which it may be introduced.

A“native” or“wild type” nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence. Thus, for example, a“wild type mRNA” is an mRNA that is naturally occurring in or endogenous to the organism. A“endogenous” nucleic acid sequence is a nucleotide sequence naturally associated with a host cell into which it is introduced.

Also as used herein, the terms“nucleic acid,”“nucleic acid molecule,”“nucleotide sequence” and“polynucleotide” refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made.

As used herein, the term“nucleotide sequence” refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, a DNA fragment or portion, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single stranded or double stranded. The terms“nucleotide sequence”“nucleic acid,”“nucleic acid molecule,”“oligonucleotide” and“polynucleotide” are also used interchangeably herein to refer to a heteropolymer of nucleotides. Nucleic acid molecules and/or nucleotide sequences provided herein are presented herein in the 5’ to 3’ direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR §§1.821 - 1.825 and the World Intellectual Property Organization (WIPO) Standard ST.25. A“5’ region” as used herein can mean the region of a polynucleotide that is nearest the 5’ end. Thus, for example, an element in the 5’ region of a polynucleotide can be located anywhere from the first nucleotide located at the 5’ end of the polynucleotide to the nucleotide located halfway through the polynucleotide. A“3’ region” as used herein can mean the region of a polynucleotide that is nearest the 3’ end. Thus, for example, an element in the 3’ region of a polynucleotide can be located anywhere from the first nucleotide located at the 3’ end of the polynucleotide to the nucleotide located halfway through the polynucleotide.

As used herein, the term "gene" refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxyribonucleotide (AMO) and the like. Genes may or may not be capable of being used to produce a functional protein or gene product. Genes can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and/or 5' and 3' untranslated regions). A gene may be "isolated" by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from

recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid.

The terms "complementary" or "complementarity," as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.

For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A."

Complementarity between two single-stranded molecules may be "partial," in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

“Complement” as used herein can mean 100% complementarity or identity with the comparator nucleotide sequence or it can mean less than 100% complementarity (e.g.,

"substantial complementarity", e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like, complementarity).

A“portion” or“fragment” of a nucleotide sequence of the invention will be understood to mean a nucleotide sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) to a reference nucleic acid or nucleotide sequence and comprising, consisting essentially of and/or consisting of a nucleotide sequence of contiguous nucleotides identical or almost identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment or portion according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent.

Different nucleic acids or proteins having homology are referred to herein as

“homologues.” The term homologue includes homologous sequences from the same and other species and orthologous sequences from the same and other species.“Homology” refers to the level of similarity between two or more nucleic acid and/or amino acid sequences in terms of percent of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties among different nucleic acids or proteins. Thus, the compositions and methods of the invention further comprise homologues to the nucleotide sequences and polypeptide sequences of this invention.“Orthologous,” as used herein, refers to homologous nucleotide sequences and / or amino acid sequences in different species that arose from a common ancestral gene during speciation. A homologue of a nucleotide sequence of this invention has a substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100%) to said nucleotide sequence of the invention.

As used herein“sequence identity” refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids.“Identity” can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

As used herein, the term“percent sequence identity” or“percent identity” refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference (“query”) polynucleotide molecule (or its complementary strand) as compared to a test (“subject”) polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments,“percent identity” can refer to the percentage of identical amino acids in an amino acid sequence.

As used herein, the phrase“substantially identical,” or“substantial identity” in the context of two nucleic acid molecules, nucleotide sequences or protein sequences, refers to two or more sequences or subsequences that have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments, sequences may be substantially identical over the entire length of the coding regions.

Furthermore, in some embodiments, substantially identical nucleotide or protein sequences perform substantially the same function (e.g., replicon proteins).

For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). An“identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more polynucleotide sequences may be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For purposes of this invention“percent identity” may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff &

Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.1 to less than about 0.001. Thus, in some embodiments of the invention, the smallest sum probability in a comparison of the test nucleotide sequence to the reference nucleotide sequence is less than about 0.001.

Two nucleotide sequences can also be considered to be substantially complementary when the two sequences hybridize to each other under stringent conditions. In some

representative embodiments, two nucleotide sequences considered to be substantially

complementary hybridize to each other under highly stringent conditions.

“Stringent hybridization conditions” and“stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I chapter 2 “Overview of principles of hybridization and the strategy of nucleic acid probe assays” Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH.

The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T_m for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences which have more than 100

complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.1 5M NaCl at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes {see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example of a medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45°C for 15 minutes. An example of a low stringency wash for a duplex of, e.g, more than 100 nucleotides, is 4-6x SSC at 40°C for 15 minutes. For short probes {e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30°C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This can occur, for example, when a copy of a nucleotide sequence is created using the maximum codon degeneracy permitted by the genetic code.

The following are examples of sets of hybridization/wash conditions that may be used to clone homologous nucleotide sequences that are substantially identical to reference nucleotide sequences of the invention. In one embodiment, a reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C. In another embodiment, the reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C. In still further embodiments, the reference nucleotide sequence hybridizes to the“test” nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, or in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 65°C.

In particular embodiments, a further indication that two nucleotide sequences or two polypeptide sequences are substantially identical can be that the protein encoded by the first nucleic acid is immunologically cross reactive with the protein encoded by the second nucleic acid.

Any nucleotide sequence and/or recombinant nucleic acid molecule of this invention can be codon optimized for expression in any organism of interest. Codon optimization is well known in the art and involves modification of a nucleotide sequence for codon usage bias using species specific codon usage tables. The codon usage tables are generated based on a sequence analysis of the most highly expressed genes for the organism/species of interest. When the nucleotide sequences are to be expressed in the nucleus, the codon usage tables are generated based on a sequence analysis of highly expressed nuclear genes for the species of interest. The modifications of the nucleotide sequences are determined by comparing the species specific codon usage table with the codons present in the native polynucleotide sequences. As is understood in the art, codon optimization of a nucleotide sequence results in a nucleotide sequence having less than 100% identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like) to the native nucleotide sequence but which still encodes a polypeptide having the same function as that encoded by the original, native nucleotide sequence. Thus, in representative embodiments of the invention, a nucleotide sequence such as SEQ ID NOs:3-8 or a polynucleotide of interest comprised in a vector of this invention may be codon optimized for expression in the particular plant species of interest.

In some embodiments, the nucleic acids, polynucleotides and polypeptides of the invention are“isolated.” An“isolated” nucleic acid, an“isolated” polynucleotide or an “isolated” polypeptide is a nucleic acid, polynucleotide or polypeptide that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid, polynucleotide or polypeptide may exist in a purified form that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the nucleic acid, polynucleotide or polypeptide. In

representative embodiments, the isolated nucleic acid, the isolated polynucleotide and/or the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more pure.

In other embodiments, an isolated nucleic acid, polynucleotide sequence or polypeptide may exist in a non-native environment such as, for example, a recombinant host cell. Thus, for example, with respect to nucleotide sequences, the term“isolated” means that it is separated from the chromosome and/or cell in which it naturally occurs. A polynucleotide is also isolated if it is separated from the chromosome and/or cell in which it naturally occurs in and is then inserted into a genetic context, a chromosome and/or a cell in which it does not naturally occur (e.g., a different host cell, different regulatory sequences, and/or different position in the genome than as found in nature). Accordingly, the nucleic acid, polynucleotides and their encoded polypeptides are“isolated” in that, by the hand of man, they exist apart from their native environment and therefore are not products of nature, however, in some embodiments, they can be introduced into and exist in a recombinant host cell.

By“operably linked” or“operably associated” as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related. Thus, the term“operably linked” or“operably associated” as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated. Thus, a first nucleic acid that is operably linked to a second nucleotide sequence, means a situation when the first nucleic acid is placed in a functional relationship with the second nucleic acid. For instance, a promoter is operably associated with a polynucleotide if the promoter effects the transcription or expression of said nucleotide sequence. Those skilled in the art will appreciate that the control sequences ( e.g ., promoter) need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a polynucleotide, and the promoter can still be considered“operably linked” to the nucleotide sequence.

As used herein,“contact”, contacting”,“contacted,” and grammatical variations thereof, refers to placing the components of a desired reaction together under conditions suitable for carrying out the desired reaction (e.g., transcriptional control, genome editing, nicking, cleavage, and/or amplifying nucleic acids).

Any plant (or groupings of plants, for example, into a genus or higher order

classification) can be employed in practicing this invention including an angiosperm, a

gymnosperm, a monocot, a dicot, a C3, C4, CAM plant, a microalgae, and/or a macroalgae. Thus, for example, types of plants useful with this invention may include woody, herbaceous, horticultural, agricultural, forestry, nursery, ornamental plant species and plant species useful in the production of biofuels, and combinations thereof.

The term“plant part,” as used herein, includes but is not limited to reproductive tissues (e.g., petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower bud, ovules, seeds, embryos, nuts, kernels, ears, cobs and husks); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, shoots, branches, bark, apical meristem, axillary bud, cotyledon, hypocotyls, and leaves); vascular tissues (e.g., phloem and xylem); specialized cells such as epidermal cells, parenchyma cells, chollenchyma cells, schlerenchyma cells, stomates, guard cells, cuticle, mesophyll cells; callus tissue; and cuttings. The term“plant part” also includes plant cells, including plant cells that are intact in plants and/or parts of plants, plant protoplasts, plant tissues, plant organs, plant cell tissue cultures, plant calli, plant clumps, and the like. As used herein,“shoot” refers to the above ground parts including the leaves and stems. As used herein, the term“tissue culture” encompasses cultures of tissue, cells, protoplasts and callus.

As used herein,“plant cell” refers to a structural and physiological unit of the plant, which typically comprise a cell wall but also includes protoplasts. A plant cell of the present invention can be in the form of an isolated single cell or can be a cultured cell or can be a part of a higher-organized unit such as, for example, a plant tissue (including callus) or a plant organ.

In some embodiments, a plant cell can be an algal cell. In some embodiments of this invention, a plant, plant part or plant cell can be from a genus including, but not limited to, the genus of Camelina, Sorghum, Gossypium, Brassica, Allium, Armoracia, Poa, Agrostis, Lolium, Festuca, Calamogrostis, Deschampsia, Spinacia,

Beta , Pisum, Chenopodium, Helianthus, Pastinaca, Daucus, Petroselium, Populus, Prunus, Castanea, Eucalyptus, Acer, Quercus, Salix, Juglans, Picea, Pinus, Abies, Lemna, Wolffla, Spirodela, Oryza or Gossypium.

In additional embodiments, the plant, plant part or plant cell can be, but is not limited to, a plant of, or a plant part, or plant cell from wheat, barley, oats, turfgrass (bluegrass, bentgrass, ryegrass, fescue), feather reed grass, tufted hair grass, spinach, beets, chard, quinoa, sugar beets, lettuce, sunflower ( Helianthus annuus ), peas {Pisum sativum), parsnips {Pastinaca sativa), carrots {Daucus carotd), parsley {Petroselinum crispum), duckweed, pine, spruce, fir, eucalyptus, oak, walnut, or willow. In some embodiments, a plant and/or part thereof useful with the invention may include, but is not limited to, arabidopsis, apple, tomato, pear, pepper {Capsicum), bean (e.g., green and dried), cucurbits (e.g., squash, cucumber, honeydew melon, watermelon, cantaloupe, and the like), papaya, mango, pineapple, avocado, stone fruits (e.g., plum, cherry, peach, apricot, nectarine, and the like), grape (wine and table), strawberry, raspberry, blueberry, mango, cranberry, gooseberry, banana, fig, citrus (e.g., clementine, kumquat, orange, grapefruit, tangerine, mandarin, lemon, lime, and the like), nuts (e.g., hazelnut, pistachio, walnut, macadamia, almond, pecan, and the like), lychee {Litchi), soybeans, corn, sugar cane, camelina, peanuts, cotton, canola, oilseed rape, sunflower, rapeseed, alfalfa, timothy, tobacco, tomato, sugarbeet, potato, pea, carrot, cereals (e.g., wheat, rice, barley, rye, millet, sorghum, oat, triticale, and the like), buckwheat, quinoa, turf, lettuce, roses, tulips, violets, basil, oil palm, elm, ash, oak, maple, fir, spruce, cedar, pine, birch, cypress, coffee, miscanthus, arundo, and/or switchgrass.

In nature, the eccDNA replicon confers the ability to a plant to sustain lethal amounts of the commonly used herbicide, glyphosate by way of massive gene amplification. The eccDNA replicon is a highly dynamic nucleic acid unit that evolved naturally in Palmer amaranth as an adaptive survival mechanism to rapidly increase gene copy number of the EPSPS gene in response to the herbicide glyphosate. Overexpression of the gene and its product of translation in Palmer amaranth confers resistance to glyphosate. While not wishing to be limited to any particular theory, the genesis of the replicon may be connected to repeated glyphosate applications. However, the replicon is stable and persists across generations without fitness penalty to the plant, and may be transferred to related plant species through hybridization. Copy numbers of the EPSPS replicon in resistant Palmer amaranth may be 100-fold higher than in sensitive plants.

The eccDNA replicon is a segment of DNA having a size of about 399 kb (SEQ ID

NO:20), which is present in a circular form in the extra-nuclear space in resistant pigweed, and contains 59 predicted genes with functional signatures that may endow critical cellular processes necessary for stress avoidance, maintenance, stability, replication, and tethering of the eccDNA replicon. Encoded in the replicon are single copies of two prominent genes one for EPSPS and another for a reverse transcriptase. Expression of the reverse transcriptase is greater by about four times that of EPSPS. The amplicon contains many other gene fragments and repeat sequences. The EPSPS replicon sequence in resistant plants is not contiguous in the sensitive plants, indicating that the cassette is the product of fast adaptive evolution, derived from repeated transpositions over time. Natural hybridization between Palmer amaranth and spiny amaranth has resulted in transfer of the extra nuclear replicon into hybrid offspring with expression of glyphosate resistance traits, which indicates the unit is heritable and compatible between species.

The eccDNA element is a large, about 399kb, plasmid-like structure that can exist outside of the chromosomes (termed herein as the eccDNA replicon).We have identified and sequenced this element to reveal very unique findings such as the putative ability to

autonomously replicate and the discovery of an encoded copy of the EPSPS gene and other transcriptionally active genes that may be involved in extreme detoxification, transport, stress avoidance, and recombination. Interestingly, the presence and maintenance of the eccDNA replicon does not seem to endow a fitness penalty, suggesting a crucial evolved biological role. In fact, this unit may even endow a fitness boost, or even confer a global increase in abiotic stress resilience. Furthermore, we have found that the eccDNA replicon is stable across generations, and traits (glyphosate resistance) can be transferred to other plant species by hybridization (e.g., Amaranthus palmeri and Amaranthus spinosa ), suggesting potential breakthrough uses as new ways to transfer and amplify DNA for trait enhancement. The repetitive content and the clustered palindromes that flank the EPSPS gene are also likely to yield additional structural and functional insights.

The present invention is directed to constructs utilizing elements of“eccDNA replicon” or“eccDNA element” in unique and non-natural configurations as a circular vector for use in genome modification in plants. Thus, in some embodiments, constructs or vectors of the invention may be provided as extranuclear plasmids engineered to amplify or introduce genes and biochemical pathways of choice into plants of interest, such as crop plants. Thus, in some embodiments, a plant vector of this invention may be tethered to the genome and not integrate into the genome, tethered or associated with the genome similar to the eccDNA element (Fig.1, panel A). In some embodiments, a plant vector of this invention may be integrated into the chromosome (Fig.1, panel B). In some embodiments, a plant vector of this invention is circular and self-replicating.

Polynucleotides suitable for use with this invention include those associated with association or tethering of extra-circularDNA (eccDNA) molecules to the nuclear chromosomes as a means to provide genomic persistence during cell division and to maintain the claimed circular vector in the plant germ line. Such polynucleotides may also include histone-binding and non-histone binding DNA association protein complexes and those that encode reporter polypeptides ( e.g ., an enzyme), including but not limited to, Green Fluorescent Protein, Red Fluorescent Protein, b-galactosidase, luciferase, alkaline phosphatase, and/or b -glucuronidase (GUS) as well as polynucleotides comprising origin of replication that can functions to facilitate target gene copy amplification.

Thus, in some embodiments, the present invention provides a circular plant vector comprising a first tethering nucleic acid and a second tethering nucleic acid; a nucleic acid encoding a polynucleotide of interest (POI); a nucleic acid comprising an origin of replication; and two or more nucleic acids encoding replicon proteins.

The term "replicon protein" as used herein, for the constructs of the invention means any protein that assists in the replication, tethering and maintenance (e.g., maintenance in the germ line of the plant, plant part or plant cell) of the vector and the POI (transgene).

In some embodiments, a first tethering nucleic acid may comprises a sequence having at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identity) to the nucleotide sequence of SEQ ID NO:l; the second tethering nucleic acid comprises a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:2;the nucleic acid comprising an origin of replication comprises a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:9; and the two or more nucleic acids encoding replicon proteins comprises sequences having at least 80% sequence identity (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identity) to any one or more of the nucleotide sequences of SEQ ID NO:3-8, in any combination. In some embodiments, a circular plant vector of this invention may comprise, 5' to 3', the first tethering nucleic acid, a first nucleic acid encoding a replicon protein; the second tethering nucleic acid, a second nucleic acid encoding a replicon protein, and the nucleic acid comprising an origin of replication. In some embodiments, the circular plant vector may further comprise, 5 'to 3', a third nucleic acid encoding a replicon protein, a fourth nucleic acid encoding a replicon protein, a fifth nucleic acid encoding a replicon protein, a sixth nucleic acid encoding a replicon protein, and a seventh nucleic acid encoding a replicon protein which are located between the second nucleic acid encoding a replicon protein and the nucleic acid comprising an origin of replication.

In some embodiments, a circular plant vector of the invention comprises, 5' to 3', a first tethering nucleic acid having the nucleotide sequence of SEQ ID NO:l; a nucleic acid encoding a polynucleotide of interest (POI); a first nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NOs:3-8; a second tethering nucleic acid having the nucleotide sequence of SEQ ID NO:2; a third nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NOs:3-8; a fourth nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NOs:3-8; a fifth nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NOs:3- 8; a sixth nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NOs:3-8; a seventh nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NOs:3-8; and a nucleic acid comprising an origin of replication having the nucleotide sequence of SEQ ID NO: 9.

In some embodiments, a circular plant vector of the invention may comprise, 5' to 3', a first tethering nucleic acid having the nucleotide sequence of SEQ ID NO:l; a nucleic acid encoding a polynucleotide of interest (POI); a first nucleic acid encoding a replicon protein having the nucleotide sequence of SEQ ID NO:3 [AP_R.00g000200]; a second tethering nucleic acid having the nucleotide sequence of SEQ ID NO:2; a second nucleic acid encoding a replicon protein having the nucleotide sequence of SEQ ID NO:3 [AP_R.00g000250]; a third nucleic acid encoding a replicon protein having the nucleotide sequence of SEQ ID NO:4

[AP_R.00g000493]; a fourth nucleic acid encoding a replicon protein having the nucleotide sequence of SEQ ID NO:5 [AP_R.00g000494]; a fifth nucleic acid encoding a replicon protein having the nucleotide sequence of SEQ ID NO:6 [AP_R.00g000430]; a sixth nucleic acid encoding a replicon protein having the nucleotide sequence of SEQ ID NO:7

[AP_R.00g000496]; a seventh nucleic acid encoding a replicon protein having the nucleotide sequence of SEQ ID NO:8 [AP_R.00g000450]; and a nucleic acid comprising an origin of replication having the nucleotide sequence of SEQ ID NO: 9. In some embodiments, the one or more nucleic acids of the vector of this invention, e.g., the first tethering nucleic acid, the second tethering nucleic acid, the nucleic acid encoding a polynucleotide of interest (POI), the nucleic acid comprising an origin of replication, and/or the two or more nucleic acids encoding replicon proteins, may be linked to one another directly or may be linked via one or more linkers (e.g, 1, 2, 3, 4, 5, or more), or any combination thereof.

In some embodiments, two or more linker polynucleotides may be used in tandem to separate the nucleic acids comprised in the vector of the invention, e.g., the first tethering nucleic acid, the second tethering nucleic acid; the nucleic acid encoding a polynucleotide of interest (POI); the nucleic acid comprising an origin of replication; and/or the two or more nucleic acids encoding replicon proteins.

A linker for use in separating two or more of the nucleic acids of the vector of the invention may be composed of any set of consecutive nucleotides that allow the replication of the vector and expression of the POI. In some embodiments, a linker useful with this invention may have a length in a range from about 10 nucleotides to about 120 nucleotides or more (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,

60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,

86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,

109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 or more nucleotides in length, or more or any range or value therein; e.g., about 10 nucleotides to about 40 nucleotides, about 10 nucleotides to about 50 nucleotides, about 10 nucleotides to about 80 nucleotides, about 10 nucleotides to about 100 nucleotides, about 20 nucleotides to about 50 nucleotides, about 20 nucleotides to about 70 nucleotides, about 20 nucleotides to about 80 nucleotides, about 20 nucleotides to about 100 nucleotides, about 20 nucleotides to about 110 nucleotides, about 20 nucleotides to about 120 nucleotides, about 30 nucleotides to about 50 nucleotides, about 30 nucleotides to about 60 nucleotides, about 30 nucleotides to about 80 nucleotides, about 30 nucleotides to about 100 nucleotides, about 30 nucleotides to about 110 nucleotides, about 30 nucleotides to about 120 nucleotides, about 40 nucleotides to about 80 nucleotides, about 40 nucleotides to about 100 nucleotides, about 40 nucleotides to about 110 nucleotides, about 40 nucleotides to about 120 nucleotides, about 50 nucleotides to about 100 nucleotides, about 50 nucleotides to about 110 nucleotides, or about 50 nucleotides to about 120 nucleotides). In some embodiments, a linker useful with this invention may comprise, for example, a nucleotide sequence any one or more of SEQ ID NO:10, SEQ ID NO:ll, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and/or SEQ ID NO: 19.

When more than one linker (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more linkers) is used to link the nucleic acids of a vector of the invention, the linkers may all be the same linker or the linkers may different from one another, or any combination thereof (e.g., some linkers may be the same as one another and others may be different).

In some embodiments, a circular plant vector of the invention may comprise a first tethering nucleic acid linked via a first linker to a nucleic acid encoding a polynucleotide of interest (POI); the nucleic acid encoding a polynucleotide of interest (POI) linked via a second linker to a first nucleic acid encoding a replicon protein; the first nucleic acid encoding a replicon protein linked via third linker to a second tethering nucleic acid; the second tethering nucleic acid linked via a fourth linker to a second nucleic acid encoding a replicon protein; the second nucleic acid encoding a replicon protein linked via a fifth linker to a third nucleic acid encoding a replicon protein; the third nucleic acid encoding a replicon protein linked directly to a fourth nucleic acid encoding a replicon protein; the fourth nucleic acid encoding a replicon protein linked via a sixth linker to a fifth nucleic acid encoding a replicon protein; the fifth nucleic acid encoding a replicon protein linked via a seventh linker to a sixth nucleic acid encoding a replicon protein; the sixth nucleic acid encoding a replicon protein linked via an eighth linker to a seventh nucleic acid encoding a replicon protein; the seventh nucleic acid encoding a replicon protein linked via a ninth linker to a nucleic acid comprising an origin of replication; and the nucleic acid comprising an origin of replication is linked via an tenth linker to the first tethering nucleic acid. In some embodiments, a linker that allows a vector of this invention to replicate and the polynucleotide of interest comprised there to be expressed may be any nucleotide sequence having a length of about 10 nucleotides to aboutl20 nucleotides. In some embodiments, each of the first linker, second linker, third linker, fourth linker, fifth linker, sixth linker, seventh linker, eighth linker, ninth linker, and tenth linker comprise a nucleotide sequence of any one of SEQ ID NOs: 10-19, in any combination, or each of the linkers may comprise any two or more of the nucleotide sequences of any one of SEQ ID NOs:10-19, in any combination (e.g., two or more linker polynucleotides in tandem). In some embodiments, the first linker is SEQ ID NO:10, the second linker is SEQ ID NO:ll, the third linker is SEQ ID NO:12 or SEQ ID NO:13, the fourth linker is SEQ ID NO:10 or SEQ ID NO:ll, the fifth linker is SEQ ID NO:12 or SEQ ID NO:14, the sixth linker is SEQ ID NO:15, the seventh linker is SEQ ID NO:16, the eighth linker is SEQ ID NO:17 or SEQ ID NO:18, the ninth linker is SEQ ID NO: 19 and the tenth linker is any one or more of the nucleotide sequences of

SEQ ID NOs: 10-19.

A circular plant vector of the invention may comprise one or more polynucleotides of interest (POIs) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more POI). A polynucleotide of interest may be any polynucleotide that imparts a desirable agronomic trait to the plant into which it is introduced. A polynucleotide of interest may encode a polypeptide that imparts a desirable agronomic trait to the plant or may confer such traits as male sterility, or may improve fertility and/or nutritional quality. Other suitable polypeptides include enzymes that degrade organic pollutants or remove heavy metals. Such plants, and the enzymes that can be isolated therefrom, are useful in methods of environmental protection and remediation. Alternatively, the heterologous nucleotide sequence can encode a therapeutically or pharmaceutically useful polypeptide or an industrial polypeptide (e.g., an industrial enzyme). Therapeutic polypeptides include, but are not limited to antibodies and antibody fragments, cytokines, hormones, growth factors, receptors, enzymes and the like.

Additional non-limiting examples of polynucleotides of interest that are suitable for use with this invention (e.g., to be expressed in response to exposure to nitrate, drought, and/or rehydration) include polynucleotides associated with nutrient uptake including transport and assimilation of organic and inorganic nutrients. Thus, for example, polynucleotides encoding polypeptides involved in nitrogen transport and assimilation, including but not limited to, nitrite transporter (NiTRl gene), high affinity nitrate transporter, nitrate and chloride transporter, nitrate reductase (nr2), NADH-dependent nitrate reductase, oligopeptide and nitrate transporter, ammonium transporter (Osamtl.l; 1.3; 2.2; 3.1; 5.1), nitrate transporter (Atntl 1), symbiotic ammonium transporter, ammonium transporter, NADH-dependent glutamate synthase, nitrate transporter, ammonium transporter (Osamtl.l; 5.2), high affinity nitrate transporter (nar2.1), gln4, gl5, nitrate transporter (nrtl.l), amino acid transport protein, NADH-dependent nitrate reductase (nrl), nitrate transporter (nrtl-5), ammonium transporter (Osamt2.1; 2.3; 3.3), high affinity nitrate transporter (nar2.1; nar2.2), nitrate transporter (Glycine max nrtl.2), ferredoxin- dependent glutamate synthase, and/or high affinity nitrate transporter (nrt2.1)

Other non-limiting examples of polynucleotides of interest include those involved in resistance to insects, nematodes and pathogenic diseases. Such may encode polypeptides that include, but are not limited to, glucosinolates (defense against herbivores), chitinases or glucanases and other enzymes which destroy the cell wall of parasites, ribosome-inactivating proteins (RIPs) and other proteins of the plant resistance and stress reaction as are induced when plants are wounded or attacked by microbes, or chemically, by, for example, salicylic acid, jasmonic acid or ethylene, or lysozymes from nonplant sources such as, for example, T4- lysozyme or lysozyme from a variety of mammals, insecticidal proteins such as Bacillus thuringiensis endotoxin, a-amylase inhibitor or protease inhibitors (cowpea trypsin inhibitor), lectins such as wheatgerm agglutinin, RNAses or ribozymes. Further non-limiting examples include nucleic acids which encode the Trichoderma harzianum chit42 endochitinase (GenBank Acc. No.: S78423) or the N-hydroxylating, multi-functional cytochrome P-450 (CYP79) protein from Sorghum bicolor (GenBank Acc. No.: U32624), or functional equivalents of these, chitinases, for example from beans (Brogue et al. (1991) Science 254:1194-1197),

“polygalacturonase-inhibiting protein” (PGIP), thaumatine, invertase and antimicrobial peptides such as lactoferrin (Lee T J et al. (2002) JAmer Soc Horticult Sci 127(2): 158-164) (See, e.g. , U.S. Patent No. 8,071,749) as well as the plant defense genes, including but not limited to, PR1, BG2, PR5, and NPR1 (or NIM1).

Also useful with the present invention are polynucleotides encoding polypeptides involved in plant hormone production or signaling including, but not limited to, auxins, cytokinins, gibberellins, strigolactones, ethylene, jasmonic acid, and brassinosteroids, as well as other polynucleotide and polypeptide sequences that regulate or effect root and leaf growth and development. Non-limiting examples of such polynucleotide and/or polypeptide sequences include GA-Deficient-1 (GA1; CPS), Gibberellin 20-Oxidase (GA20ox, GA5 (in At)),

Gibberellin 2-beta-dioxygenase (GA2ox), Gibberellin 3 -Oxidase (GA3ox), GA-Insensitive (GAI),GA Regulated MYB(GAMYB), GCA2 Growth Controlled By ABA 2 (GCA2), G-Protein Coupled Receptor (GCR1), Glycosyl Hydrolase Family-45 (GH45), tryptophan synthase alpha chain (e.g.,GRMZM2G046163, GRMZM2G015892), Auxin Binding Protein 1 (ABP1), IAA- amino acid hydrolase ILR1 (e.g., GRMZM2G091540), phosphoribosylanthranilate transferase, Indole Acetic Acid 17/Auxin Resistant 3(IAA17, AXR3), Indole Acetic Acid 3/Short Hypocotyl (IAA3, SHY2), IAA-lysine synthetase (iaaL), tryptophan monooxygenase (iaaM), IAA-Aspartic Acid Hydrolase (IaaspH), IAA-Glucose Synthase (IAGLU),IndoleAcetamide Hydrolase (IAH), Indole-3 -Acetaldehyde Oxidase (IAO),IAA-ModifiedProtein (IAP1), Auxin Response factors (ARFs), small auxin up RNA (SAUR), Induced By Cytokinin 6 (Same as ARR5)(IBC6), Induced By Cytokinin 7 (Same as ARR4) IBC7, Viviparous-14 ( Vpl4 ), PLA2 (Zhu J-K. Annual Review of Plant Biology 2002, 53(l):247-273), ATPLC2 (Benschop et al. Plant Physiology 2007, 143(2): 1013-1023), inositol polyphosphate 5-phosphatase (At5PTaseI), calcium- dependent protein kinases (CDPKs), calcineurin B-like (CBL) calcium sensor protein

CBL4/SOS3, CIPK-like protein 1, ACC (1-aminocyclopropane-1-carboxylate) synthase, ACC oxidase, phosphatase 2C ABI1, TINY, maize lipoxygenase 7 (GRMZM2G070092), allene oxide synthase (AOS) (e.g., GRMZM2G033098 and GRMZM2G376661), short chain alcohol dehydrogenases (ADH), Tasselseed2 ( Ts2 ), Tasselseedl ( Tsl ), Super centipede 1

( Scn1/GDIl,e.g AT2G44100), RDH2 (Carol et al. Nature 2005, 438(7070):1013-1016.), G- signaling proteins, Morphogenesis of Root Hair (MRH), AtAGC2-1 (e.g., At3g25250), Cellulose Synthase-Like D3 (CSLD3), xylosyltransferase 2 (e.g., At4g02500, AtXX2), xyloglucan endotransglucosylase/hydrolase 26 (e.g., AtXTH26, At4g28850), xyloglucan

endotransglycosylase, xyloglucan galact-osyltransferase (MUR3 (e.g.,AT2G20370), ARP2/3 (WURM/DISTORTED 1) complex, and germin-like protein (e.g., AT5G39110).

Other polynucleotides and polypeptides suitable for use with the present invention include those that confer the“stay-green” phenotype (See, Hortensteiner, S. Trends in Plant Science 14: 155-162 (2009)). Non-limiting examples of such nucleotide sequences include MtSGR, MsSGR (Zhou et al. Plant Physiol. 157: 1483-1496 ( 2011)), STAY-GREEN (SGR or SGN) (Jiang et al, Plant J 52: 197-209 (2007)), Park et al, Plant Cell 19: 1649-1664 (2007)), NONYELLOWING ( NYE1 ) (Ren et al, Plant Physiol 144: 1429-1441 (2007)), and/or GREEN- FLESH (GF) or CHLOROPHYLL RETAINER (CL) (Barry et al., Plant Physiol 147: 179-187 (2008)).

Polynucleotides involved in grain filling are also useful with the present invention and include, but are not limited to GIF I (GRAIN INCOMPLETE FILLING 1 ) from rice.

Other non-limiting examples of polynulcoetides of interest that are suitable for production in plants include those resulting in agronomically important traits such as herbicide resistance (also sometimes referred to as“herbicide tolerance”), virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, and/or fungal resistance. See, e.g., U.S. Patent Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431. The

polynucleotide also can be one that confers increased plant vigor or yield (including traits that allow a plant to grow at different temperatures, soil conditions and levels of sunlight and precipitation), or one that allows identification of a plant exhibiting a trait of interest (e.g., a selectable marker, seed coat color, etc.). Various polynucleottides of interest, as well as methods for introducing these polypeptides into a plant, are described, for example, in U.S. Patent Nos. 4,761,373; 4,769,061; 4,810,648; 4,940,835; 4,975,374; 5,013,659; 5,162,602; 5,276,268; 5,304,730; 5,495,071; 5,554,798; 5,561,236; 5,569,823; 5,767,366; 5,879,903, 5,928,937; 6,084,155; 6,329,504 and 6,337,431; as well as US Patent Publication No.

2001/0016956. See also, on the World Wide Web at

lifesci . sussex. ac .uk/home/N eil_Crickmore/Bt/.

Polynucleotides conferring resistance/tolerance to an herbicide that inhibits the growing point or meristem, such as an imidazalinone or a sulfonylurea can also be suitable in some embodiments of the invention. Exemplary nucleotide sequences in this category code for mutant ALS and AHAS enzymes as described, e.g., in U.S. Patent Nos. 5,767,366 and

5,928,937. U.S. Patent Nos. 4,761,373 and 5,013,659 are directed to plants resistant to various imidazalinone or sulfonamide herbicides. U.S. Patent No. 4,975,374 relates to plant cells and plants containing a nucleic acid encoding a mutant glutamine synthetase (GS) resistant to inhibition by herbicides that are known to inhibit GS, e.g., phosphinothricin and methionine sulfoximine. U.S. Patent No. 5,162,602 discloses plants resistant to inhibition by

cyclohexanedione and aryloxyphenoxypropanoic acid herbicides. The resistance is conferred by an altered acetyl coenzyme A carboxylase (ACCase).

In some embodiments, a polynucleotide may increase tolerance of a plant, plant part and/or plant cell to heat stress and/or high temperature. The polynucleotide may encode a polypeptide or inhibitory polynucleotide (e.g., functional RNA) that results in increased tolerance to heat stress and/or high temperature. Suitable polynucleotides include without limitation polynucleotides encoding water stress polypeptides, ABA receptors, and dehydration proteins (e.g., dehydrins (ERDs)).

In some embodiments, polynucleotides that encode polypeptides that provide tolerance to water stress (e.g., drought) may be incorporated into a vector of this invention and

transformed into a plant as described herein. Non-limiting examples of polypeptides that provide tolerance to water stress include: water channel proteins involved in the movement of water through membranes; enzymes required for the biosynthesis of various osmoprotectants (e.g., sugars, pro line, and Glycine-betaine); proteins that protect macromolecules and membranes (e.g., LEA protein, osmotin, antifreeze protein, chaperone and mR A binding proteins); proteases for protein turnover (thiol proteases, Clp protease and ubiquitin); and detoxification enzymes (e.g., glutathione S -transferase, soluble epoxide hydrolase, catalase, superoxide dismutase and ascorbate peroxidase). Non-limiting examples of proteins involved in the regulation of signal transduction and gene expression in response to water stress include protein kinases (MAPK, MAPKKK, S6K, CDPK, two-component His kinase, Bacterial-type sensory kinase and SNF1); transcription factors (e.g., MYC and bZIP); phosopholipase C; and 14-3-3 proteins.

Polynucleotide sequences that encode receptors/binding proteins for abscisic acid (ABA) are also useful in the practice of the present invention. Non-limiting examples of ABA binding proteins/receptors include: the Mg-chelatase H subunit; RNA-binding protein FCA; G-protein coupled receptor GCR2; PYR1; PYL5; protein phosphatases 2C ABI1 and ABI2; and proteins of the RCAR (Regulatory Component of the ABA Receptor) family.

In some embodiments, a polynucleotide sequence may encode a dehydration protein, also known as a dehydrin ( e.g ., an ERD). Dehyration proteins are a group of proteins known to accumulate in plants in response to dehydration. Examples include WCOR410 from wheat; PCA60 from peach; DHN3 from sessile oak, COR47 from Arabidopsis thaliana Hsp90, BN59, BN115 and BnerdlO from Bras sic a napus COR39 and WCS19 from Triticum aestivum (bread wheat); and COR25 from Brassica rapa subsp. Pekinensis. Other examples of dehydration proteins are ERD proteins, which include without limitation, ERDl, ERD2, ERD4, ERD5, ERD6, ERD8, ERD 10, ERD11, ERD13, ERD15 and ERD 16.

Polynucleotide sequences conferring resistance to glyphosate are also suitable for use with the present invention. See, e.g., U.S. Patent No. 4,940,835 and U.S. Patent No. 4,769,061. U.S. Patent No. 5,554,798 discloses transgenic glyphosate resistant maize plants, which resistance is conferred by an altered 5-enolpyruvyl-3-phosphoshikimate (EPSP) synthase gene. Heterologous polynucleotides suitable to confer tolerance to the herbicide glyphosate also include, but are not limited to the Agrobacterium strain CP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Patent No. 5,633,435 or the glyphosate oxidoreductase gene (GOX) as described in U.S. Patent No. 5,463,175. Other heterologous polynucleotides include genes conferring resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., mutant forms of the acetolactate synthase (ALS) gene that lead to such resistance, in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides that act to inhibit the action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene). The bar gene encodes resistance to the herbicide basta, the nptll gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS gene encodes resistance to the herbicide chlorsulfuron.

Polynucleotide sequences coding for resistance to phosphono compounds such as glufosinate ammonium or phosphinothricin, and pyridinoxy or phenoxy propionic acids and cyclohexones are also suitable. See, European Patent Application No. 0 242 246. See also, U.S. Patent Nos. 5,879,903, 5,276,268 and 5,561,236.

Other suitable polynucleotides of interest include those coding for resistance to herbicides that inhibit photosynthesis, such as a triazine and a benzonitrile (nitrilase). See, U.S. Patent No. 4,810,648. Additional suitable nucleotide sequences coding for herbicide resistance include those coding for resistance to 2,2-dichloropropionic acid, sethoxydim, haloxyfop, imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine herbicides, s-triazine herbicides and bromoxynil. Also suitable are polynucleotide sequences conferring resistance to a protox enzyme, or that provide enhanced resistance to plant diseases; enhanced tolerance of adverse environmental conditions (abiotic stresses) including but not limited to drought, heat stress, high temperature, cold, excessive soil salinity or extreme acidity or alkalinity; and alterations in plant architecture or development, including changes in developmental timing.

See, e.g., U.S. Patent Publication No. 2001/0016956 and U.S. Patent No. 6,084,155.

Insecticidal proteins useful in the invention may be produced in an amount sufficient to control insect pests, i. e. , insect controlling amounts. It is recognized that the amount of production of insecticidal protein in a plant useful to control insects may vary depending upon the cultivar, type of insect, environmental factors and the like. Suitable heterologous

polynucleotides that confer insect tolerance include those which provide resistance to pests such as rootworm, cutworm, European Corn Borer, and the like. Exemplary nucleotide sequences include, but are not limited to, those that encode toxins identified in Bacillus organisms (see, e.g., WO 99/31248; U.S. Patent Nos. 5,689,052; 5,500,365; 5,880,275); Bacillus thuringiensis toxic protein genes (see, e.g., U.S. Patent Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; 6,555,655; 6,541,448; 6,538,109; Geiser, et al. (1986) Gene 48:109); and lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825). Polynucleotide sequences encoding Bacillus thuringiensis (Bt) toxins from several subspecies have been cloned and recombinant clones have been found to be toxic to lepidopteran, dipteran and coleopteran insect larvae (for example, various delta-endotoxin genes such as CrylAa, Cry 1 Ah, Cry 1 Ac, Cry IB, Cry 1C, CrylD, CrylEa, CrylFa, Cry 3 A, Cry9A, Cry9C and Cry9B; as well as genes encoding vegetative insecticidal proteins such as Vipl, Vip2 and Vip3). A full list of Bt toxins can be found on the worldwide web at Bacillus thuringiensis Toxin Nomenclature Database maintained by the University of Sussex (see also, Crickmore et al. (1998) Microbiol. Mol. Biol. Rev.

62:807-813).

Polynucleotides encoding polypeptides that are suitable for production in plants further include those that improve or otherwise facilitate the conversion of harvested plants and/or plant parts into a commercially useful product, including, for example, increased or altered

carbohydrate content and/or distribution, improved fermentation properties, increased oil content, increased protein content, improved digestibility, and increased nutraceutical content, e.g., increased phytosterol content, increased tocopherol content, increased stand content and/or increased vitamin content. Polynucleotides of interest may also include, for example, those resulting in, or contributing to, a reduced content of an unwanted component in a harvested crop, e.g., phytic acid, or sugar degrading enzymes. By“resulting in” or“contributing to” is intended that the polynucleotide of interest can directly or indirectly contribute to the existence of a trait of interest (e.g., increasing cellulose degradation by the use of a heterologous cellulase enzyme).

In some embodiments, a polynucleotide of interest may contribute to improved digestibility for food or feed. Xylanases are hemicellulolytic enzymes that improve the breakdown of plant cell walls, which leads to better utilization of the plant nutrients by an animal. This leads to improved growth rate and feed conversion. Also, the viscosity of the feeds containing xylan can be reduced by xylanases. Heterologous production of xylanases in plant cells also can facilitate lignocellulosic conversion to fermentable sugars in industrial processing.

Numerous xylanases from fungal and bacterial microorganisms have been identified and characterized (see, e.g., U.S. Patent No. 5,437,992; Coughlin et al. (1993)“Proceedings of the Second TRICEL Symposium on Trichoderma reesei Cellulases and Other Hydrolases” Espoo; Souminen and Reinikainen, eds. (1993) Foundation for Biotechnical and Industrial

Fermentation Research 8:125-135; U.S. Patent Publication No. 2005/0208178; and PCT Publication No. WO 03/16654). In particular, three specific xylanases (XYL-I, XYL-II, and XYL-III) have been identified in T. reesei (Tenkanen et al. (1992) Enzyme Microb. Technol. 14:566; Torronen et al. (1992) Bio/Technology 10:1461; and Xu et al. (1998) Appl. Microbiol. Biotechnol. 49:718).

In some embodiments, a polynucleotide useful with the present invention can be a polysaccharide degrading enzyme. Plants producing such an enzyme may be useful for generating, for example, fermentation feedstocks for bioprocessing. In some embodiments, enzymes useful for a fermentation process include alpha amylases, proteases, pullulanases, isoamylases, cellulases, hemicellulases, xylanases, cyclodextrin glycotransferases, lipases, phytases, laccases, oxidases, esterases, cutinases, granular starch hydrolyzing enzyme or other glucoamylases.

Polysaccharide-degrading enzymes include: starch degrading enzymes such as alpha- amylases (EC 3.2.1.1), glucuronidases (E.C. 3.2.1.131), exo-1,4-alpha-D glucanases such as amyloglucosidases and glucoamylase (EC 3.2.1.3), beta-amylases (EC 3.2.1.2), alpha- glucosidases (EC 3.2.1.20), and other exo-amylases, starch debranching enzymes, such as a) isoamylase (EC 3.2.1.68), pullulanase (EC 3.2.1.41), and the like; b) cellulases such as exo-1,4- 3-cellobiohydrolase (EC 3.2.1.91), exo-1,3-beta-D-glucanase (EC 3.2.1.39), beta-glucosidase (EC 3.2.1.21); c) L-arabinases, such as endo-1,5-alpha-L-arabinase (EC 3.2.1.99), alpha- arabinosidases (EC 3.2.1.55) and the like; d) galactanases such as endo-1,4-beta-D-galactanase (EC 3.2.1.89), endo-1,3-beta-D-galactanase (EC 3.2.1.90), alpha-galactosidase (EC 3.2.1.22), beta-galactosidase (EC 3.2.1.23) and the like; e) mannanases, such as endo-1,4-beta-D- mannanase (EC 3.2.1.78), beta-mannosidase (EC 3.2.1.25), alpha-mannosidase (EC 3.2.1.24) and the like; f) xylanases, such as endo-1,4-beta-xylanase (EC 3.2.1.8), beta-D-xylosidase (EC 3.2.1.37), 1,3-beta-D-xylanase, and the like; and g) other enzymes such as alpha-L-fucosidase (EC 3.2.1.51), alpha-L-rhamnosidase (EC 3.2.1.40), levanase (EC 3.2.1.65), inulanase (EC 3.2.1.7), and the like.

Further polynucleotides that encode enzymes which may be used with the present invention include those that encode proteases, such as fungal and bacterial proteases. Fungal proteases include, but are not limited to, those obtained from Aspergillus, Trichoderma, Mucor and Rhizopus, such as A. niger, A. awamori, A. oryzae and M. miehei.

Other useful enzymes include, but are not limited to, hemicellulases, such as mannases and arabinofuranosidases (EC 3.2.1.55); ligninases; lipases (e.g., E.C. 3.1.1.3), glucose oxidases, pectinases, xylanases, transglucosidases, alpha 1,6 glucosidases (e.g., E.C. 3.2.1.20);

cellobiohydrolases; esterases such as ferulic acid esterase (EC 3.1.1.73) and acetyl xylan esterases (EC 3.1.1.72); and cutinases (e.g. E.C. 3.1.1.74).

A polynucleotide of interest may also encode a reporter polypeptide (e.g., an enzyme) or selectable marker, including but not limited to Green Fluorescent Protein, b-galactosidase, luciferase, alkaline phosphatase, the GUS gene encoding b -glucuronidase, and chloramphenicol acetyltransferase. Further examples of selectable markers include, but are not limited to, a nucleotide sequence encoding aadA (i.e., spectinomycin and streptomycin resistance), a nucleotide sequence encoding neo (i.e., kanamycin resistance), a nucleotide sequence encoding aphA6 (i.e., kanamycin resistance), a nucleotide sequence encoding nptll ( i.e., kanamycin resistance), a nucleotide sequence encoding bar (i.e., phosphinothricin resistance), a nucleotide sequence encoding cat (i.e., chloramphenicol resistance), a nucleotide sequence encoding badh (i.e., betaine aldehyde resistance), a nucleotide sequence encoding egfp, (i.e., enhanced green fluorescence protein), a nucleotide sequence encoding gfp (i.e., green fluorescent protein), a nucleotide sequence encoding luc (i.e., luciferase), a nucleotide sequence encoding mCherry (i.e. a red fluorescent protein), a nucleotide sequence encoding ble ( bleomycin resistance), a nucleotide sequence encoding ereA (erythromycin resistance), and any combination thereof.

Additional examples of selectable markers useful with the invention include, but are not limited to, a nucleotide sequence encoding an altered 5-enolpyruvylshikimate-3 -phosphate (EPSP) synthase, which confers resistance to glyphosate (Hinchee et al. (1988) Biotech. 6:915- 922); a nucleotide sequence encoding a nitrilase such as bxn from Klebsiella ozaenae that confers resistance to bromoxynil (Stalker et al. (1988) Science 242:419-423); a nucleotide sequence encoding an altered acetolactate synthase (ALS) that confers resistance to

imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP Patent Application No. 154204); a nucleotide sequence encoding a methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chem. 263:12500-12508); a nucleotide sequence encoding a dalapon dehalogenase that confers resistance to dalapon; a nucleotide sequence encoding a mannose-6-phosphate isomerase (also referred to as phosphomannose isomerase (PMI)) that confers an ability to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629); a nucleotide sequence encoding an altered anthranilate synthase that confers resistance to 5- methyl tryptophan; and/or a nucleotide sequence encoding hph that confers resistance to hygromycin.

Additional selectable markers include, but are not limited to, a nucleotide sequence encoding b-lactamase, an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75:3737- 3741); a nucleotide sequence encoding xylE that encodes a catechol dioxygenase (Zukowsky et al. (1983 ) Proc. Natl. Acad. Sci. USA 80:1101-1105); a nucleotide sequence encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form melanin (Katz et al. (1983) J. Gen. Microbiol. 129:2703-2714); a nucleotide sequence encoding b-galactosidase, an enzyme for which there are chromogenic substrates; a nucleotide sequence encoding luciferase (lux) that allows for bioluminescence detection (Ow et al. (1986) Science 234:856-859); a nucleotide sequence encoding Bla that confers ampicillin resistance; or a nucleotide sequence encoding aequorin which may be employed in calcium- sensitive bioluminescence detection (Prasher et al. (1985) Biochem. Biophys. Res. Comm.

126:1259-1268), and/or any combination thereof. One of skill in the art is capable of choosing a suitable selectable marker for use with the vector of this invention.

In some embodiments, a vector of the invention may comprise CRISPR-Cas structure and machinery for genome integration (e.g., DNA palindromes, helicase domain, integrase domain, nuclease, reverse transcriptase).

Where appropriate, a polynucleotide of interest may be optimized for increased expression in a transformed plant, e.g. , by using plant preferred codons. Methods for synthetic optimization of nucleic acid sequences are available in the art. The nucleotide sequence of interest can be optimized for expression in a particular host plant or alternatively can be modified for optimal expression in monocots. See, e.g., EP 0 359 472, EP 0 385 962, WO 91/16432; Perlak et al, Proc. Natl. Acad. Sci. USA 88, 3324 (1991), and Murray et al, Nuc. Acids Res. 17, 477 (1989), and the like. Plant preferred codons can be determined from the codons of highest frequency in the proteins expressed in that plant. Methods

Further provided are methods of using the vectors of the invention. In some

embodiments, the invention provides a method of expressing a polynucleotide of interest in a plant or part thereof, the method comprising: introducing into the plant or part thereof a circular plant vector of the invention, and selecting a plant or part thereof comprising the circular plant vector and expressing the polynucleotide of interest.

In some embodiments, a method of modulating the expression a polynucleotide of interest in a plant cell is provided, the method comprising introducing into a plant cell a circular plant vector of the invention to produce a transformed plant cell comprising the circular plant vector and expressing the polynucleotide of interest. In some embodiments, the plant cell is a population of plant cells and following introduction of the vector, cells are selected from the population that comprise the vector and express the polynucleotide of interest.

In some embodiments, a method of producing a plant cell expressing a polynucleotide of interest is provided, the method comprising introducing into a plant cell a circular plant vector of the invention, thereby producing a plant cell comprising the circular plant vector and expressing the polynucleotide of interest. In some embodiments, the plant cell is a population of plant cells and following introduction of the vector, cells are selected from the population that comprise the vector and express the polynucleotide of interest.

In some embodiments, the methods of the invention may further comprise regenerating a plant from the plant part comprising the circular plant vector and expressing the polynucleotide of interest. In some embodiments, the methods of the invention may additionally comprise regenerating a plant from the plant cell comprising the circular plant vector and expressing the polynucleotide of interest to produce a plant comprising the circular plant vector and expressing the polynucleotide of interest.

In some embodiments, the methods of the invention provide a plant, plant part or plant cell that is stably transformed. In some embodiments, the vector comprising the polynucleotide of interest present in a stably transformed plant or part thereof (e.g., a cell) is tethered to the chromosome of the plant. In some embodiments, the vector or part thereof may be integrated into the genome of the plant or part thereof. In some embodiments, the vector or part thereof is not integrated into the genome of the plant or part thereof.

The invention further provides stably transformed plants and/or stably transformed plant cells or other plant parts produced by the method the invention. Also provided are seeds produced from the plants of the invention, wherein the seeds comprise the vector of the invention and polynucleotide of interest. The invention also provides products harvested from the stably transformed plants of the invention, the product comprising the vector and polynucleotide of interest. Further provided are processed products produced from harvested products from the plants of the invention including seed, the processed products comprising the vector and polynucleotide of interest.

In some embodiments, a crop comprising a plurality of the stably transformed plant of the invention is also provided. The crop may be grown, for example, in a field (e.g., a cultivated field, an agricultural field), a growth chamber, a greenhouse, a recreational area, a lawn, and/or a roadside and the like.

“Introducing,” in the context of a polynucleotide sequence of interest (e.g., a circular plant vector of this invention), means presenting the polynucleotide sequence of interest to the plant, plant part, and/or plant cell in such a manner that the polynucleotide sequence gains access to the interior of a cell. Thus, the term“transformation” as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient. Thus, in some embodiments, a plant cell of the invention is stably transformed with a vector of the invention. In other embodiments, a plant of the invention is transiently

transformed with a vector of the invention.

“Transient transformation” in the context of a polynucleotide means that a

polynucleotide is introduced into the cell and does not integrate into the genome of the cell.

By“stably introducing” or“stably introduced” in the context of a polynucleotide introduced into a cell it is intended that the introduced polynucleotide is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide.

“Stable transformation” or“stably transformed” as used herein means that a nucleic acid molecule is introduced into a cell and integrates into the genome of the cell or tethers itself to a chromosome in the cell. As such, the integrated/tethered nucleic acid molecule is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations.“Genome” as used herein also includes the nuclear and the plastid genome, and therefore includes integration/tethering of the nucleic acid into, for example, the chloroplast genome/to the chloroplast DNA. Stable transformation as used herein can also refer to a transgene that is maintained extrachromasomally, for example, as a minichromosome. The phrase“a stably transformed plant, plant part, and/or plant cell expressing said one or more polynucleotide sequences or a vector of this invention” and similar phrases used herein, means that the stably transformed plant, plant part, and/or plant cell comprises the one or more polynucleotide sequences/vector and that said one or more polynucleotide sequences/vector are functional in said stably transformed plant, plant part, and/or plant cell. In some embodimetns, a vector of the invention may be stably transformed into a plant or part thereof, e.g., plant cell, wherien the vector and/or part thereof is stably intergrated into the genome. In some

embodiments, the vector comprising the polynucleotide of interest is not incorporated into the genome of the stably transformed plant but is tethered to a chromosome and is maintained stably in the plant or part thereof (e.g., plant cell).

Transient transformation may be detected by, for example, an enzyme-linked

immunosorbent assay (ELISA) or Western blot, which can detect the presence of a peptide or polypeptide encoded by one or more transgene introduced into an organism. Stable

transformation of a cell can be detected by, for example, a Southern blot hybridization assay of genomic DNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into an organism (e.g., a plant). Stable transformation of a cell can be detected by, for example, a Northern blot hybridization assay of RNA of the cell with nucleic acid sequences which specifically hybridize with a nucleotide sequence of a transgene introduced into a plant or other organism. Stable transformation of a cell can also be detected by, e.g., a polymerase chain reaction (PCR) or other amplification reactions as are well known in the art, employing specific primer sequences that hybridize with target sequence(s) of a transgene, resulting in amplification of the transgene sequence, which can be detected according to standard methods. Transformation can also be detected by direct sequencing and/or hybridization protocols that are well known in the art.

A circular plant vector of this invention may be introduced into a cell of a plant by any method known to those of skill in the art. In some embodiments of the invention, transformation of a cell comprises nuclear transformation. In some embodiments of the invention,

transformation of a cell comprises plastid transformation.

Procedures for transforming plants are well known and routine in the art and are described throughout the literature. Non-limiting examples of transformation methods include transformation via bacterial-mediated nucleic acid delivery (e.g., via Agrobacteria), viral- mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium-phosphate-mediated transformation, cyclodextrin-mediated transformation,

electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, as well as any other electrical, chemical, physical (mechanical) and/or biological mechanism that results in the introduction of nucleic acid into the plant cell, including any combination thereof. General guides to various plant transformation methods known in the art include Miki et al. (“Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J. E., Eds. (CRC Press, Inc., Boca Raton, 1993), pages 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol.

Lett. 7:849-858 (2002)). General guides to the transformation of yeast include Guthrie and Fink (1991) (Guide to yeast genetics and molecular biology. In Methods in Enzymology, (Academic Press, San Diego) 194:1-932) and guides to methods related to the transformation of bacteria include Aune and Aachmann (Appl. Microbiol Biotechnol 85:1301-1313 (2010)).

A polynucleotide therefore can be introduced into a plant, plant part, plant cell in any number of ways that are well known in the art. The methods of the invention do not depend on a particular method for introducing one or more nucleotide sequences into a plant, only that they gain access to the interior the cell. Where more than polynucleotide is to be introduced, they can be assembled as part of a single nucleic acid construct, or as separate nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, the polynucleotide can be introduced into the cell of interest in a single transformation event, or in separate transformation events, or, alternatively, a polynucleotide can be incorporated into a plant as part of a breeding protocol.

In some embodiments, when a plant part or plant cell is stably transformed, it can then be used to regenerate a stably transformed plant comprising a vector of this invention encoding a polynucleotide of interest in or tethered to its genome. Means for regeneration can vary from plant species to plant species, but generally a suspension of transformed protoplasts or a petri plate containing transformed explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently root. Alternatively, somatic embryo formation can be induced in the callus tissue. These somatic embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and plant hormones, such as auxin and cytokinins. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.

The regenerated plants are transferred to standard soil conditions and cultivated in a conventional manner. The plants are grown and harvested using conventional procedures.

The particular conditions for transformation, selection and regeneration of a plant can be optimized by those of skill in the art. Factors that affect the efficiency of transformation include the species of plant, the target tissue or cell, composition of the culture media, selectable marker genes, kinds of vectors, and light/dark conditions. Therefore, these and other factors may be varied to determine an optimal transformation protocol for any particular plant species. It is recognized that not every species will react in the same manner to the transformation conditions and may require a slightly different modification of the protocols disclosed herein. However, by altering each of the variables, an optimum protocol can be derived for any plant species.

Further, the genetic properties engineered into the transgenic seeds and plants, plant parts, and/or plant cells of the present invention described herein can be passed on by sexual reproduction or vegetative growth and therefore can be maintained and propagated in progeny plants. Generally, maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as harvesting, sowing or tilling.

In some embodiments, plant vector of this invention is circular and self-replicating. In some embodiments, the eccDNA comprises origin of replication sites. In some embodiments, the eccDNA of a construct of the invention may comprise CRISPR-like structure and machinery for genome integration (e.g., DNA palindromes, helicase domain, integrase domain, nuclease, reverse transcriptase).

In some embodiments, one or more nucleic acids molecules of interest (e.g., genes, non- transcribe regulatory elements etc.) may be comprised in a construct of the invention comprising an eccDNA replicon for use in expression in a plant. In some embodiments, the one or more nucleic acids molecules to be introduced or overexpressed in a plant may be heterologous or endogenous to the plant in which they are introduced, thereby providing the

expression/overexpression of the nucleic acids in the target plant. In some embodiments, the one or more nucleic acid molecules comprised in the eccDNA construct may have host genome homology.

In some embodiments, a nucleic acid molecule conferring a desired trait may be inserted into the replicon adjacent to or in tandem with the EPSPS (or another selection marker), and this expression cassette may then be introduced into a plant for expression (heterologous gene) or for overexpression (endogenous gene).

Recombinant plants may be selected for glyphosate resistance or for any other selectable marker, while the gene of interest is expressed/amplified in copy number. This invention may solve the problem of transgene recalcitrance. Constructs comprising the eccDNA and one or more nucleic acid molecules of interest may be used to tailor species-specific replicons, for transgene stacking, and for exploiting precision trait enhancement with effects that may be analogous to heterosis.

The constructs of the invention may be useful for trait enhancement and molecular breeding tools to increase gene copy number and transform crops with new genes expressing desired traits through the eccDNA intermediate tethered to the genome; apart from the difficulties associated with off-target effects described with traditional genome engineering. This technology may drastically reduce the time required to enhance and/or alter crop traits.

Advantages of the constructs of the invention include that they comprise eccDNA, which as described herein provides a natural vehicle to transfer novel genes into the genome, that they provide a unique mechanism for increasing gene copy number and that nucleic acid molecules of interest (e.g., genes, etc) may be expressed outside of the genome removing site specific gene silencing issue. Additional advantages may further include reduced regulatory cost due to gene construct being plant derived, more flexibility with regard to the means of gene delivery

(integrative or tethering) and targeted gene delivery

Potential applications include gene therapy delivery, transient expression of genes in transcriptionally relevant regions of the genome, targetable vector of genetic plant

transformations and one step dose dependent trait delivery system.

The invention will now be described with reference to the following examples. It should be appreciated that these examples are not intended to limit the scope of the claims to the invention, but are rather intended to be exemplary of certain embodiments. Any variations in the exemplified methods that occur to the skilled artisan are intended to fall within the scope of the invention.

EXAMPLES

Example 1.

Selective pressures in nature potentiate genomic plasticity as a mechanism of adaptation to sustain life ¹ The predominant source of this functional diversity in eukaryotes and prokaryotes is gene copy number variation, which endows these adaptive processes . These unbalanced structural variations impart a considerable spectrum of phenotypic diversity ⁵.

However, the underlying mechanisms that give rise to gene copy proliferation are poorly understood. Here, we show a unique result of genomic plasticity, the amplification of a massive extrachromosomal circular DNA (eccDNA), which is an extra-nuclear vehicle used by the Amaranthus palmeri genome to rapidly increase crucial gene copy numbers required for plant survival under extreme abiotic stress. This functional eccDNA confers resistance to the herbicide glyphosate. Upon exposure and continued selection with glyphosate, the A. palmeri genome has undergone extensive shuffling to form a plasmid-like structure, a massive eccDNA replicon, that harbors the EPSP synthase gene and other encoded machinery whose functions traverse detoxification, replication, recombination, and membrane transport. The eccDNA can exist in the extranuclear space and may replicate autonomously to function as a vehicle for gene amplification. Furthermore, the eccDNA replicon is comprised of a complex arrangement of repeat sequences and mobile genetic elements interspersed among arrays of clustered palindromes that may be crucial for stability, DNA duplication, and/or a means of nuclear integration of the adjacent and intervening sequences. While the discovery of the eccDNA replicon may provide new approaches in understanding genome dynamics and the link between functional eccDNA and evolutionary mechanisms, it also holds potential to ignite a revolution in biotechnology and plant breeding as a new vehicle for DNA amplification and genome engineering.

McClintock ⁶ stated“a sensing mechanism must be present in plants when experiencing unfavorable conditions to alert the cell to imminent danger and to set in motion the orderly sequence of events that will mitigate this danger.” Plants, being sedentary, are subject to the prevailing conditions in which they grow. Under favorable conditions, growth ensues and gene expression supports physiological needs. However, as the quote implies, when conditions become adverse, plants may alter their physiology by activation of diverse stress-avoidance signaling cascades ⁷. One such stress avoidance/response mechanism is the amplification of the gene encoding 5-enoylpyruvylshikimate-3 -phosphate synthase ( EPSPS ), which confers resistance to the herbicide glyphosate in A. palmeri. The EPSPS gene may be amplified 40 to 100-fold in highly resistant populations ⁸. We examined the genomic architecture surrounding the EPSPS gene to discover structure, content, and putative mechanisms of DNA amplification and regulatory elements.

In glyphosate-resistant A. palmeri , previous work shows EPSPS distribution among many chromosomes, suggesting a transposon-based mechanism of mobility; while EPSP synthase activity was also elevated ⁹. Amplification of the EPSPS gene and its product, EPSP synthase, ameliorates the unbalanced or unregulated metabolic changes, such as shikimate accumulation and loss of aromatic amino acids associated with glyphosate activity in sensitive plants ¹⁰. Interestingly EPSPS amplification also correlates with significant genome expansion (e.g., 11% increase in genome size with ~100 extra copies), which includes co-amplification of many other genes, transcription factors and repetitive elements ⁸. Without wishing to be limited by any particular theory, the mechanism of amplification may be governed by a diverse array of master transcriptional regulators being activated to create novel combinations of response genes enabling the plant to circumvent the chemical assault ¹¹. Here, we present a mechanism of rapid adaptive evolution by a unique vehicle for massive gene copy amplification: The

extrachromosomal DNA (eccDNA) replicon.

Example 2. Plant Material and Fiber-FISH The glyphosate-resistant (GR) A. palmeri plants were collected in 2013 from a soybean field in Riley county, Kansas that was exposed to extensive use of glyphosate during the decade prior by Dr. Mithila Jugulam. Young leaf tissues were collected from fast growing plants of GR A. palmeri. Nuclei isolation, DNA fiber preparation, and fiber-FISH were performed following published protocols ^30,31. In total, 6 overlapping BAC clones comprise the minimal tile path of the replicon, which were analyzed in pairs by FISH on DNA fibers prepared from leaf tissue. Fiber-FISH images were captured with a Zeiss Axioplan 2 microscope using a cooled CCD camera CoolSNAP HQ2 (Photometries) and AxioVision 4.8 software (Zeiss). The final contrast of the images was processed using Adobe Photoshop CS5 software.

Example 3. BAC Isolation, Sequencing, and Analysis

BAC library construction, partial tile path isolation, sequencing and analysis were described previously.⁸ Two additional BAC clones, 08H14 and 01G15 on the eccDNA replicon ends, were determined by chromosome walking by hybridization with overgo probes designed from unique distal sequence on the terminal ends of the EPSPS cassette (clones 03 A06 and 13C09). These two BAC clones were harvested and sequenced using Pacific Biosciences RSII sequencing to a depth greater than 100X, as described in Molin et al. ⁸ Raw single molecule sequence was self-corrected using the CANU Celera assembler (Koren et al., 2017) with the corOutCoverage=1000 to increase the output of corrected sequences. BAC end sequences were determined using standard Sanger sequencing methods and aligned to the reference assemblies with Phrap and opened in Consed (Gordon et al, 1998) for editing. BAC overlaps were identified using CrossMatch (Gordon et al., 1998) and ends joined manually to form a circular structure. The consensus eccDNA replicon was annotated using a combination of the MakerP pipeline (Campbell et al., 2014) with RNAseq (below) used as evidence with final manual curation. Functional domain scans and homology based annotations were determined by BLAST, InterproScan, and HMM using the SwissProt, non-redundant, and PfamA databases, respectively. Repeat characterization and masking were conducted with the RepeatMasker software (http://www.repeatmasker.org). MITE and helitron sequences were predicted with the detectMITE (Ye et al, 2016) and HelitronScanner(Xiong et al., 2014) tools. Circular figures were prepared using the Circos plotting toolset (Krzywinski et al., 2009).

Example 4. RNAseq and Plant Material

Glyphosate-resistant (GR) A. palmeri plants were collected in 2013 by W. M from a soybean field in Washington County, Mississippi that was exposed to extensive application of glyphosate during the decade prior. Young leaf tissues (2 cm) were collected from fast growing plants of GR palmeri and used as a source for the glyphosate exposure experiment.

Glyphosate treatment experiments were conducted in a greenhouse at the Jamie Whitten Delta States Research Center of USDA-ARS in Stoneville, Mississippi set to 25/20°C ± 3^°C day/night temperature and a 15-h photoperiod under natural sunlight conditions supplemented with high- pressure sodium lights providing 400 mmol m-2 s-1. Seeds were sown on the surface of potting mix surface (Metro-Mix 360, Sun Gro Horticulture, Bellevue, WA) and lightly covered with 2 mm of mix, subirrigated and grown to the two-true leaf stage at which time they were sprayed with glyphosate solution (0.42 kg-ai-ha-l) using an air-pressurized indoor spray chamber (DeVries Manufacturing Co., Hollandale, MN) equipped with a nozzle mounted with 8002E flat-fan tip (Spraying Systems Co., Wheaton, IL) delivering 190 L-ha-1 at 220 kPa. All of the seedlings survived confirming glyphosate resistance in this 2013 population. Seedlings from this population that had not been sprayed were allowed to grow until 2.5-cm tall and were transplanted into 8 cm x 8 cm x 7 cm pots containing the same potting mix. Thereafter, plants were watered as needed and fertilized once two weeks after transplanting with a water-soluble fertilizer (Miracle-Gro, Scotts Miracle-Gro Products, Inc., Marysville, OH). When seedlings reached the six-leaf stage they were sprayed with either water, or water plus surfactant plus glyphosate using the spray chamber. The surfactant was 0.5 % v/v Tween 20 and glyphosate was applied at 0.42 kg.ai.ha-1 after neutralization with 0.1 KOH solution. Leaves from the third and fourth nodes were harvested for RNA extraction at 0, 4 and 24 hours after treatment. Plants were held for two weeks post leaf harvest to verify survival following glyphosate treatment.

Total RNA was harvested at 4 and 24h in biological triplicates using the RNeasy plant mini kit (Qiagen). Purified RNA was verified for intactness on a Bioanalyzer 2100 (Agilent) and subject to stranded mRNA-seq using standard TruSeq procedures and sequenced to a target depth of at least 15M reads per sample. Raw sequence data was preprocessed for adapter and low-quality bases with the Trimmomatic tool (Krzywinski et al., 2009) and cleaned reads aligned to the eccDNA replicon consensus assembly with Bowtie2 v.2.3.4.1(Langmead and Salzberg, 2012) and the following arguments: -no-mixed -no-discordant -gbar 1000 -end-to- end -k 200 -q -X 800. TMM and FPKM transcript quantification was determined with RSEM vl .3.0 (Li and Dewey, 2011).

Example 5. Cloning, Plasmid Construction and Yeast Transformation

The eccDNA ARS sequences were amplified from 23A10 BAC using primers

167,312F_CEN-SLIC and 168,187R_SLIC (Supplementary Table 4). The yeast vector, pRS315, was linearized via PCR using primers pRS_ACEN-F and pRS_AARS-R such that the CEN6 sequence remained, but the ARS was removed. Q5 polymerase was used for all PCRs. The eccDNA ARS was assembled into pRS305 using a SLIC reaction. Constructs were confirmed with a restriction digest and sequencing .Saccharomyces cerevisiae (ATCC 208288) were transformed as previously described ⁴⁵. Yeast cells were grown in a YPD (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) preculture overnight at 28 °C and 250 rpm. In a 250-mL baffled flask, 50 mL of pre-warmed YPD was inoculated to a final titer of 5 x 10⁶ cells/mL. The culture was grown to a final titer of 2 x 10⁷ cells/mL at 28 °C and 250 rpm. Cells were harvested by centrifugation at 3,000 x g for 5 minutes. The cell pellet was resuspended in 25 mL of sterile milliQ water and centrifuged again three times before cells were resuspended in 1.0 mL of sterile water. Cell pellet was then resuspended in 360 mL freshly made transformation mix (240 mL PEG 3350 (50% w/v), 36 mL 1.0 M LiAc, 50 mL single-stranded salmon sperm DNA (2 mg/mL), 36 mL plasmid DNA plus sterile water). Cells were heat shocked at 42 °C for 40 minutes and resuspended in 1 mL of YPD for 2 hours at 28 °C, 250 rpm. Recovered cells were plated on YSC-Leu+2% glucose plates and grown at 28 °C for 2 days and colonies counted. pRS305 lacks an ARS and served as a negative control while pRS315 contains an ARS and served as a positive control.

Example 6. Identification and Structure of the eccDNA Plant Replicon

Advancing upon our recent work ⁸, FISH identified EPSPS signals that reside in the extra-nuclear space and in a seemingly random distribution on the mitotic metaphase chromosome in the glyphosate resistant A. palmeri nucleus (Fig. 2, panel A). Detailed resolution of the extra-nuclear signals revealed a circular, plasmid-like structure (Fig. 2, panel B). Fiber-FISH analysis of contiguous BAC clones (grouped into 2 pools for red/green labels) revealed relative BAC positions (Fig. 2, panel B) and further verified circular structure, (Fig. 2, panel B). Two-color FISH of contiguous BACs also reveal clear, consistent signals that illustrate each tiled BAC clone generates a linearized FISH signal (Fig. 5). Single-molecule sequencing of the BAC tile path further corroborates the circular structure as a massive extrachromosomal DNA element, much larger than anything reported to date.

The eccDNA replicon is comprised of 399,435bp and contains 110 putative protein coding sequences, of which, 65 have expression profiles 24 hours after glyphosate exposure (Fig. 2, panel A and Table 1) in a glyphosate resistant biotype. The most transcriptionally active genes are the heat shock protein (Apr009) followed by the EPSPS gene (Apr035) and Apr073 (unknown function) (Table 1). Many of the eccDNA replicon encoded genes have functional signatures that may endow the critical cellular processes necessary for stress avoidance, maintenance, stability, and replication of the eccDNA replicon. These processes include DNA transport and mobility, molecule sequestration, hormonal control, DNA replication and repair, heat shock, transcription regulation, and nuclease activity, (Table 1).

For example, the replicon encodes 5 copies of aminotransferase-like genes (Apr 0003, 0005, 0030, 0069, 0099) that are ubiquitously expressed and crucial for proper cell division and differentiation¹⁴. A heat shock protein (Apr009) is a member of the Hsp70 family, which is upregulated by heat stress and toxic chemicals; and may also inhibit apoptosis¹⁵. There are 7 copies of protein suppressor of gene silencing genes that are required for post-transcriptional gene silencing, natural virus resistance, and the production of trans-acting siRNAs ’ (RNAi machinery). A gene with a NAC containing domain (Apr0084) was predicted, which represents a class of transcription factors that regulate plant defense¹⁸ and abiotic stress responses¹⁹.

Intriguingly, AprlOl harbors a SWIM domain which typically associates with RAD51 paralogs to promote homologous recombination^20,21. Apr0102 contains a helicase domain, and Apr0105 harbors domains with endo/exo-nuclease and phosphatase activity. Apr 107 contains a zinc binding reverse transcriptase domain and an integrase catalytic core, which are characteristic of a retroviral mechanism to integrate viral DNA into the host. This integrase is also found in various transposase proteins and is a member of the ribonuclease H-like superfamily involved in replication, homologous recombination, DNA repair, transposition, and RNA interference

(Table 1).

EccDNAs are an understudied fraction of the genomes of plants, humans, and other eukaryotic organisms, and their contribution to genomic expansion, genetic diversity, and ultimately the phenome is not well understood. Furthermore, there is little evidence regarding the mechanisms by which eccDNAs may function, replicate, integrate into the host genome, or segregate during cell division. The presence of such a large eccDNA, compared to the 5.7 kb eccDNA in plants , ~20kb in Drosophilia , and the ~38 kb elements in yeast ’

, indicates a unique mechanism of genome plasticity and specialized purpose to increase gene copy abundance to survive a harsh abiotic stress.

Autonomous replication

The eccDNA replicon is heavily punctuated with sharp changes in A+T and G+C content, which may imply biological function , including replication initiation sites (Fig. 3, panel A). Autonomous replicating sequences (ARS) can function as origins of replication in eukaryotes²⁸, and plants have been shown to have conserved ARS structures and sequence features commonly found in yeast and higher animals²⁹. A motif scan of the eccDNA replicon revealed a single exact match to the Extended Autonomous Consensus Sequence (EACS, 17bp), previously described in yeast and other eukaryotes²⁸ (Fig. 3, panel B). Nearby, we identified a 9bp DNA Unwinding Element (DUE) ³⁰, which is adjacent to a region with elevated A+T content (approximately 73%) in a 40bp window (20bp +/-) that has a high propensity for bending (data not shown), which can implicate an origin of replication region ³¹. By cloning +/- lkb regions containing the putative origin of replication into a selectable ARS-less yeast vector, we observed dividing colonies, verifying that the eccDNA replicon ARS sequence is functional and can facilitate replication. Recombinant yeast growth was much slower with a lower abundance of colonies on plates with the eccDNA replicon, relative to the control ARS suggesting a possible role of cis -elements and trans- factors for efficiency in the plant . For example, the eccDNA replicon encodes a DNA helicase (Apr_102), and replicon protein complexes (Apr_045), whose likely functions are required for genome stability, DNA recombination, repair, and replication. To our knowledge, no eccDNA reported as of this writing has been verified to contain a functional origin of replication, suggesting a selective advantage.

Repetitive elements and structural organization

The eccDNA replicon contains retroelements composed of SINEs, LINEs, and LTR elements, in addition to DNA transposons interspersed by predicted MITE and Helitron elements (Fig. 4, panel A, B). Flanking the EPSPS gene is an asymmetric set of direct repeats that are composed of arrays of clustered long and short interspersed palindromic repeat sequences (CLiSPrs), separated by identical MITES (Fig. 4, panel A). The complete palindromic array is bordered by LTR/ERVK, DN A/MULE MUDR and DNA/TdMar

Stowaway elements (not shown). The CLiSPr block regions are also composed of elevated A+T segments (up to 80%) (Fig. 4, panel A), which may serve as a mechanism for stability or nuclear recognition sites for tethering, integration into open chromatin, or transcriptional hotspots. Downstream of the CLiSPr arrays are repetitive triplicate clusters of LTR-Cassandra and DNA hAT-Ac elements, each bordered by MITE elements. Still further downstream are clustered A-rich and LTR/Gypsy elements. These clustered arrangements may indicate functional relationships. The LTR/Cassandra and LTR/Gypsy elements divide the coding regions other than that contained within the direct repeats into 3 segments from 195 to 250 kb, from 287 to 355kb and from 395 to 90 kb. The EACS and DUE sites are located at 287 kb just after the LTR/Cassanra repeats which is appropriately positioned to initiate transcription of transcribed genes.. The eccDNA replicon is a unique result of genomic plasticity and a massive eccDNA vehicle for gene amplification and expression of genes outside of the nuclear chromatin. The origin is unknown, but likely a result of mobile element activation and extensive A. palmeri genome shuffling invoked by heavy abiotic pressures. It has various functional modalities for integration, replication, stability and maintenance to ensure survival. Furthermore, because of the functional implications of the putative genes in the eccDNA replicon, the presence of this unit may also contribute a fitness boost and correlate with a general increase in abiotic stress resilience, or perhaps, an increased disposition to adapt. This vehicle may afford new directions in breeding and biotechnology through deeper understanding of genome interaction and integration, targeted gene amplification, and by transfer of novel pathways into new genomes through an engineered eccDNA intermediate.

Genome tethering

The first cytological evidence of an eccDNA tethering to chromosomes in plants as a mechanism of genome persistence was recently reported (Koo et al., Plant Physiol 176, 1932- 1938 (2018)). EccDNA maintenance has been extensively studied in DNA viruses that maintain their genomes as extrachromosomal circular DNAs, such as Epstein-Barr, Rhadinovirus, papillomavirus, and others (Feeney and Parish, Proc Biol Sci 276, 1535-1544 2009). A commonality among these viruses is genome tethering, which is facilitated by virus encoded DNA-binding proteins that associate with repeated sequences in the viral genome that also have an affinity with host cell proteins that associate with mitotic chromatin to ensure nuclear retention (Feeney and Parish, 2009). For example, the Epstein-barr virus has been reported to anchor to the host genome through interaction of encoded EBNA1 and the cellular protein EBP2 forming a protein-protein interaction or by directly associating to chromatin via an AT-hook motif that binds to A/T rich sequences on metaphase chromosomes (Sears et al., J Virol 78,

11487-11505, 2004), (Wu et al. Nature 575, 699-703, 2000). In Rhadinoviruses, a role has been suggested for the gene LANA, which is thought to interact with terminal repeat regions (TR) in the virus (comprised of long tandem repeats) (Russo et al. Proc Natl Acad Sci U S A 93, 14862- 14867, 1996), where the C-terminus of the protein attaches the TR and the N-terminus tethers the episome to the chromosome (Piolot et al. J Virol 75, 3948-3959, 2001). In Papillomaviruses, the E2 gene is a multifunctional DNA-binding protein that interacts with the El helicase for replication (Masterson et al. J Virol 72, 7407-7419, 1998) and facilitates genome association through interaction within the N-terminal transactivation domain (Skiadopoulos and McBride J Virol 72, 2079-2088, 1998). Computational analysis of the eccDNA replicon revealed several genes which may function in the tethering mechanism. AP_R.00g000496 contains 2 core AT- hook motifs (GRP) and also encodes a zinc finger SWIM domain which is recognized to bind DNA, proteins, and/or lipid structures. The optimal binding sequences of the core AT-hook are AAAT and AATT, which when bound together forms a concave DNA conformation for tight binding(Reeves Environ Health Persp 108, 803-809, 2000). In the eccDNA replicion, there are 143 and 186 (AAAT)2 or (AATT)2 motifs, respectively. There are consistent clusters of tandemly repeated motifs in the CLiSPr repeats. These AT-hook gene products may interact with the A/T rich regions of the eccDNA replicon and other nuclear scaffold proteins.

Furthermore, the helicase domain has recently been demonstrated to be an important regulator of the chromatin association, establishment, and maintenance of the Herpes virus.

AP_R.00g000496 is predicted to encode a helicase motif which may also have a role in tethering of the eccDNA to nuclear chromatin.

The eccDNA replicon is a massive eccDNA vehicle for gene amplification, trait expression, maintenance and transfer of genomic information. This is the first report of an autonomously replicating functional plant eccDNA. The origin is unknown, but likely a result of mobile element activation and extensive genome shuffling invoked by heavy abiotic pressures. It has various functional modalities for integration, replication, stability and maintenance to ensure survival. Furthermore, because of the functional implications of the putative genes in the eccDNA replicon, the presence of this unit could cause a general increase in abiotic stress resilience, or perhaps, an increased disposition to adapt. This vehicle affords new directions in breeding and biotechnology through deeper understandings of its origin and function.

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The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

THAT WHICH IS CLAIMED IS:

1. A circular plant vector comprising:

a first tethering nucleic acid and a second tethering nucleic acid;

a nucleic acid encoding a polynucleotide of interest (POI);

a nucleic acid comprising an origin of replication; and

two or more nucleic acids encoding replicon proteins.

2. The circular plant vector of claim 1, wherein

the first tethering nucleic acid comprises a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:l;

the second tethering nucleic acid comprises a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:2;

the nucleic acid comprising an origin of replication comprises a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:9; and

the two or more nucleic acids encoding replicon proteins comprise sequences having at least 90% sequence identity to any one or more of the nucleotide sequences of SEQ ID NO:3-8, in any combination.

3. The circular plant vector comprising of claim 1 or claim 2, wherein the vector comprises 5' to 3',:

the first tethering nucleic acid, a first nucleic acid encoding a replicon protein; the second tethering nucleic acid, a second nucleic acid encoding a replicon protein, and the nucleic acid comprising an origin of replication.

4. The circular plant vector comprising of claim 3, wherein the vector further comprises between the second nucleic acid encoding a replicon protein and the nucleic acid comprising an origin of replication, 5'to 3', a third nucleic acid encoding a replicon protein, a fourth nucleic acid encoding a replicon protein, a fifth nucleic acid encoding a replicon protein, a sixth nucleic acid encoding a replicon protein, and a seventh nucleic acid encoding a replicon protein.

5. The circular plant vector of claim 3 or claim 4, comprising 5' to 3',

the first tethering nucleic acid having the nucleotide sequence of SEQ ID NO:l; the nucleic acid encoding a polynucleotide of interest (POI); the first nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NOs:3-8;

the second tethering nucleic acid having the nucleotide sequence of SEQ ID NO:2; the third nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NOs:3-8;

the fourth nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NOs:3-8;

the fifth nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NOs:3-8;

the sixth nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NOs:3-8;

the seventh nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NOs:3-8; and

the nucleic acid comprising an origin of replication having the nucleotide sequence of

SEQ ID NO: 9.

6. The circular plant vector of any one of claims 3-5, comprises, 5' to 3',

the first tethering nucleic acid having the nucleotide sequence of SEQ ID NO:l; the nucleic acid encoding a polynucleotide of interest (POI);

the first nucleic acid encoding a replicon protein having the nucleotide sequence of

SEQ ID NO:3;

the second tethering nucleic acid having the nucleotide sequence of SEQ ID NO:2; the second nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NO:3;

the third nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NO: 4;

the fourth nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NO:5;

the fifth nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NO:6;

the sixth nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NO: 7;

the seventh nucleic acid encoding a replicon protein having the nucleotide sequence of any one of SEQ ID NO:8; and the nucleic acid comprising an origin of replication having the nucleotide sequence of

SEQ ID NO: 9.

7. The circular plant vector of any one of claims 2-6, wherein the vector further comprises one or more linkers for linking at least two of SEQ ID NOs:l-9.

8. The circular plant vector of claim 7 or claim 8, wherein the one or more linkers have a length in a range of about 10 nucleotides to about 100 nucleotides.

9. The circular plant vector of claim 7 or claim 8, wherein the one or more linkers are the same or are different from one another, or any combination thereof.

10. The circular plant vector of any one of claims 7-9, wherein

the first tethering nucleic acid is linked via a first linker to the nucleic acid encoding a polynucleotide of interest (POI);

the nucleic acid encoding a polynucleotide of interest (POI) is linked via a second linker to the first nucleic acid encoding a replicon protein;

the first nucleic acid encoding a replicon protein is linked via third linker to the second tethering nucleic acid;

the second tethering nucleic acid is linked via a fourth linker to the second nucleic acid encoding a replicon protein;

the second nucleic acid encoding a replicon protein is linked via a fifth linker to the third nucleic acid encoding a replicon protein;

the third nucleic acid encoding a replicon protein is linked directly to the fourth nucleic acid encoding a replicon protein;

the fourth nucleic acid encoding a replicon protein is linked via a sixth linker to the fifth nucleic acid encoding a replicon protein;

the fifth nucleic acid encoding a replicon protein is linked via a seventh linker to the sixth nucleic acid encoding a replicon protein;

the sixth nucleic acid encoding a replicon protein is linked via an eighth linker to the seventh nucleic acid encoding a replicon protein;

the seventh nucleic acid encoding a replicon protein is linked via a ninth linker to the nucleic acid comprising an origin of replication; and the nucleic acid comprising an origin of replication is linked via an tenth linker to the first tethering nucleic acid.

11. The circular plant vector of claim 10, wherein each of the first linker, second linker, third linker, fourth linker, fifth linker, sixth linker, seventh linker, eighth linker, ninth linker, and tenth linker are selected from the group of nucleotide sequences of SEQ ID NOs: 10-19, in any combination.

12. The circular plant vector of claim 10 or claim 11, wherein first linker is SEQ ID

NO:10, the second linker is SEQ ID NO:ll, the third linker is SEQ ID NO:12 or SEQ ID NO:13, the fourth linker is SEQ ID NO:10 or SEQ ID NO:ll, the fifth linker is SEQ ID NO:12 or SEQ ID NO:14, the sixth linker is SEQ ID NO:15, the seventh linker is SEQ ID NO:16, the eighth linker is SEQ ID NO:17 or SEQ ID NO:18, the ninth linker is SEQ ID NO: 19 and the tenth linker is any one of SEQ ID NOs: 10-19.

13. The circular plant vector of any one of claims 1-12, wherein the polynucleotide of interest comprises two or more polynucleotides of interest.

14. A method of expressing a polynucleotide of interest in a plant or part thereof, the method comprising:

introducing into the plant or part thereof the circular plant vector of any one of claims 1-13, and

selecting a plant or part thereof expressing the polynucleotide of interest.

15. A method of modulating the expression a polynucleotide of interest in a plant cell, the method comprising

introducing into a plant cell the circular plant vector of any one of claims 1-13 to produce a transformed plant cell expressing the polynucleotide of interest.

16. A method of producing a plant cell expressing a polynucleotide of interest, the method comprising:

introducing into a plant cell the circular plant vector of any one of claims 1-13, thereby producing a plant cell comprising the polynucleotide of interest.

17. The method of any one of claims 14-16, further comprising regenerating a plant from the plant part expressing the polynucleotide of interest of claim 13 or from the plant cell expressing the polynucleotide of interest of claim 14 or claim 15 to produce a plant expressing the polynucleotide of interest.

18. The method of any one of claims 14-17, wherein the plant, plant part or plant cell is stably transformed.

19. The method of any one of claims 14-18, wherein the vector comprising the polynucleotide of interest is tethered to the chromosome of the plant.

20. A stably transformed plant produced by the method of any one of claims 17-19.

21. A stably transformed plant cell produced by the method of claim 16 or claim 17.

22. A seed of the plant of claim 20, wherein the seed comprises the vector comprising the polynucleotide of interest.

23. A product harvested from the stably transformed plant of claim 20, the product comprising the vector comprising the polynucleotide of interest.

24. A processed product produced from the seed of claim 22 or the harvested product of claim 23, the processed product comprising the vector comprising the polynucleotide of interest.

24. A crop comprising a plurality of the stably transformed plant of claim 20.